JP2815773B2 - Asymmetric porous membrane - Google Patents

Abstract

The invention relates to the use of an asymmetrically porous membrane for the simultaneous distributed enrichment of a substance which is not adsorbed onto the membrane and which, in the form of a membrane-wetting solution, is contacted with the membrane on the fine-pore side of the membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION This invention relates to the use of asymmetric porous membranes in analytical methods and to test carriers for performing such methods.

[0002]

BACKGROUND OF THE INVENTION The term "test carrier" is used especially for blood, plasma,
A solid analytical agent that holds reagents necessary for analyzing biological liquids such as serum and urine, particularly a flat strip-shaped or slide-shaped analytical agent. The carrier material itself is a solid, which generally also includes a solid reagent. Known carrier materials include:
For example, fibrous structures such as paper, fleece, fabric, knit, nets, films that are soluble or swellable in the liquid to be tested, or porous membranes. The reagents necessary to perform the determination of the analyte in a fluid, such as a bodily fluid, may be impregnated with the carrier material in a suitable solution, or coated with a suitable spreadable paste containing the reagents and then dried. Can be manufactured by Alternatively, of course, the necessary reagents can be added during the production of the carrier material. For example, films or membranes can be made from moldable solutions or suspensions that already contain the necessary reagents for analyte determination.

[0003] When the liquid to be tested is applied to such a test carrier, the reaction between the analyte to be tested in the sample solution and the reagents present in the carrier material is carried on or in the test carrier. Happens. The reaction product is measured and represents the amount of the analyte in the sample solution to be analyzed. For example, US Pat. No. 3,607,093 discloses that at least a portion thereof is solid,
That is, a test carrier for analyzing a biological fluid, which comprises a membrane permeable to a fluid and contains a diagnostic reagent as a dry substance, is disclosed. The membrane itself is manufactured so that at least the surface portion of the membrane is impermeable to large particles such as red blood cells. It is described in the experimental part that a solution of the reagents necessary for analyzing a biological fluid is impregnated into a membrane and dried. Upon application of the liquid to be analyzed and then, if desired, wiping off the excess liquid, a change in the color of the membrane is observed if there is an analyte to be measured in the sample liquid.

[0004] EP-A-0 345 781
The number relates to a test carrier for analyzing liquids. The test carrier includes an asymmetric porous membrane that holds one or more reagents that produce a detectable substance in the presence of the analyte to be measured. To measure an analyte in a liquid, the sample solution is applied to the surface of a membrane with large pores, and the measurement is made from the surface with small pores. “BTS asymmetric membrane”, available from Filtrite (San Diego, Calif., USA), is particularly convenient because cellular blood components are separated and the reagent / analyte reaction proceeds across the membrane. In selecting a reagent that produces a detectable substance by reaction with the analyte to be measured, properties important for detection, such as color and chemiluminescence, are important if the reagent is sufficiently stable in the membrane.

[0005] European Patent Application No. 0407800 relates to test strips for the analysis of substances in biological fluids. This includes an asymmetric porous membrane preferably made from a polymer solution containing 1 to 4% by weight of an anionic surfactant. This membrane holds the reagents necessary for analyte measurement. To perform the analysis, the liquid to be tested is applied to the side of the large hole. The reaction products are measured from the small pore side of the membrane.

[0006]

Any document in the art is abundant in detectable substances (whether it is colored or chemiluminescent) on one surface of an asymmetric porous membrane having, by definition, different porosity. There is no mention of what to do. Surprisingly, when a substance in the form of a membrane-wetting solution comes into contact with the membrane, and when this substance is not or substantially not adsorbed on the membrane,
It has now been discovered that this material is concentrated and uniformly dispersed on the small pore side of an asymmetric porous membrane.

[0007]

SUMMARY OF THE INVENTION Accordingly, the present invention is directed to the use of an asymmetric porous membrane to uniformly disperse and enrich material on the small pore side of the membrane, wherein the material is adsorbed on the membrane. Is not or is not substantially adsorbed and is brought into contact with the membrane in the form of a membrane-wetting solution.

The invention also features contacting the membrane-wetting solution of the substance with an asymmetric porous membrane such that the substance is not adsorbed or substantially not adsorbed.
It relates to a method of accumulating the substance by dispersing it uniformly on one side of the membrane. Finally, the invention also relates to the use of one of the above-mentioned methods and the contacting of a solution of the substance to be measured with an asymmetric porous membrane, or the method in which the substance to be measured in a sample is present on the membrane. Formed by more than one substance,
Or released and measured from the side of the small hole,
A method for measuring a substance in a liquid sample using a test carrier, wherein the method does not allow the substance to be measured to be adsorbed on a membrane,
Alternatively, it is not substantially adsorbed.

In a particularly advantageous embodiment of the invention, the substance to be measured is hemoglobin, and a suitable test carrier is
The above method, comprising an asymmetric porous membrane that does not adsorb or substantially does not adsorb hemoglobin, and retains a hemolytic substance or an analogue thereof as an additional layer on its surface. . The term "asymmetric porous" is a convenient term commonly known in the art (see, for example, EP 0407800 or EP 03455781). It is generally understood that this is a polymer film that is porous across both sides, with the pores on one surface being larger than the pores on the other. The asymmetric porous membrane of the present invention preferably has an asymmetry factor of 10 or more, particularly preferably 100 or more.
The asymmetry factor in this case represents the ratio of the pore size on the surface of the large pores to the pore size on the surface of the small pores. According to the invention, the pore size on the side of the smaller pore is 0.0
03-3 μm asymmetric porous membranes are preferred.

Asymmetric porous membranes are described, for example, in US Pat.
774,039, U.S. Pat. No. 4,629,563,
And EP-A-0 345 781 and the like. Asymmetric porous membranes can be manufactured by one of ordinary skill in the art. Asymmetric porous membranes are commercially available, for example, Memtec Timoniu
Company B (Maryland, USA) BTS25 membrane is particularly useful for the present invention. This membrane is a porous polysulfone membrane. For example, European Patent Application Publication No. 033
No. 6483, a polyethersulfone membrane which is an alloy with polyvinylpyrrolidone was also found to be useful in practicing the present invention. Such a membrane, for example,
X-Flow B. V. It is commercially available from the company (Enshed, The Netherlands) under the trade names PS11 and PS21.

For use in the present invention, the asymmetric porous membrane must be wettable with a liquid containing the substance to be concentrated on the side of the small pores. Therefore, in the case of aqueous liquids such as body fluids such as blood, plasma, serum, and urine,
The membrane must be sufficiently hydrophilic. If the material of the membrane itself is not sufficiently hydrophilic, the polymer membrane can be made hydrophilic. For this purpose, the membrane can be treated, for example, with a substance which swells in water but is insoluble therein. For example, it is known from US Pat. No. 4,413,074 that hydroxyalkylcellulose can be used to make polymer membranes hydrophilic. Polyvinylpyrrolidone can also be a hydrophilizing agent.

In general, when a single drop of a sample solution having a volume of 20 μl is absorbed by a piece of membrane of 15 × 15 mm or more within 20 seconds at room temperature and retained in the membrane, or when asymmetric porous A membrane is considered sufficiently wettable when applying a drop of liquid to the large pore side of the membrane wets the membrane over the entire thickness of the application surface. According to the invention, any wettable membrane can be used in which the substance to be concentrated on the side of the small pores is not adsorbed.
Whether a particular membrane-substance combination meets this purpose can be determined by simple tests. For this, the substances to be tested are dissolved in a solution in which the concentration effect is to be achieved, in the case of aqueous solutions, for example, in water. The solution is passed through one or more layers of the membrane under consideration, and then tested for changes in the concentration of the substance in the solution before and after passing through the membrane. The method is carried out in the case of a colored substance as follows: A five-layer asymmetric porous membrane is fixed on a membrane filter holder without glass filter (for example, purchased from Sartorius of Göttingen, Germany), Is placed on the suction bottle with the large hole side of the membrane of each layer facing up. The substance solution to be tested is added to the large hole side of the membrane in the filter holder and vacuum is drawn through the membrane. After passing through the membrane, the absorbance of the liquid is measured with a photometer and the measured value is compared with the measured value of the original solution. If the absorbance of the solution before and after passing through the membrane is significantly different, adsorption of the dissolved substance to the membrane has occurred. The absorbance of the solution after passing through the membrane is at least about 1 less than the absorbance of the original solution.
When 5% less, it is considered significantly different. The following limiting conditions must be observed: substance concentration in solution: about 1-100 mg / l; quantity of liquid to be aspirated: about 10 ml; number of membrane discs: 5; membrane in membrane filter holder Disc diameter: about 60 mm; thickness of each membrane disc: about 110-150 μm; and speed of the suction step: about 0.25 ml / sec.

[0013] No or substantially no adsorption on the asymmetric porous membrane (15% in the test model described above)
Below) the material is substantially concentrated on the small pore side of the asymmetric porous membrane. FIG. 4 shows the enrichment profiles corresponding to FIGS. 1, 2 and 3. FIG. Examples 2 and 3 illustrate these figures in more detail. The enrichment profile showing the behavior of the material on the asymmetric porous membrane adsorbed on the membrane and not enriched on the small pore side of the membrane is shown in FIG. This example will be described in more detail in Example 10.

To concentrate the substance so that the substance is evenly distributed on the small pore side of the asymmetric porous membrane, a membrane-wetting solution of the substance is contacted with the asymmetric porous membrane according to the invention. To do this, it is convenient to immerse the membrane in the solution or to add the solution to the membrane. When the solution is added to the membrane, it does not matter whether the solution is added to the small pore side or the large pore side to obtain a concentration effect.

The above described method of concentrating a substance essentially in a uniformly dispersed form on one side of the membrane can be used for analyte carrier-bound assays. For example, a solution of the substance to be measured is brought into contact with an asymmetric porous membrane that can be wetted with the solution and measured on the membrane from the side of the small pores. As already mentioned above, the present invention requires that the substance to be measured is not adsorbed or substantially not adsorbed on the membrane. Generally, a solution of the substance to be measured is added to the large pore side of the membrane. However, the substance to be measured can be released or formed by a reagent located in or on the membrane or by a substance arranged as one or more layers on the membrane. Test carrier compositions known in the art can be used for such carrier-bound assays, but in accordance with the present invention, whether visible, optical, or reflectance photometric, should be measured. The layer is an asymmetric porous membrane as described above.

The assay according to the invention is particularly suitable for colored analytes. If the substance to be measured is not itself colored, it can be converted to a colored substance by an appropriate reaction known in the field of analytical clinical chemistry, or a colored reaction product that is indicative of the analyte to be measured. Can be produced in a chemical reaction. It is advantageous for the asymmetric porous membranes used in the present invention to have low wet transparency when optically measured by visual or instrumental measurements, particularly by reflectance photometry. That is, the reflection coefficient of the film used particularly advantageously in the present invention must be greater than 20% after wetting with the solvent of the solution to be measured,
It is even more advantageous if it is larger than. In this context, polysulfone membranes, such as BTS membranes obtained from Memtec Timmonium (Maryland, USA) are particularly advantageous for the measurement of aqueous solutions such as body fluids such as blood, plasma, serum, urine and the like. Was found.

When measuring liquid samples containing colored particle components, it has been found to be particularly advantageous to use a combination of the filter properties of an asymmetric porous membrane with low wet transparency. Therefore, the corresponding asymmetric porous membranes of the present invention are very advantageously used for the carrier-bound assay of substances in whole blood. The pore size on the small pore side of the asymmetric porous membrane is about 0.003 to 3 μm, and whole blood is applied to the large pore side of a moderately hydrophilic membrane having an asymmetry factor of 10 or more, preferably 100 or more. In addition, red blood components (red blood cells) cannot reach the small pore side of the membrane. Red blood cells are separated from plasma and serum which transport the lysed sample components and, if desired, the lysing reagents and corresponding reaction products to the small pore side of the membrane by capillary action. If the substance to be measured, which is originally present in the sample or released or formed on the membrane in contact with the sample solution and the reagent, is not adsorbed or substantially not adsorbed on the membrane material, In the course of performing this method, it is concentrated and evenly distributed on the sides of the small pores.

Thus, it is possible to perform a measurement with higher sensitivity than when there is no concentration or accumulation effect. The measurement can be performed with a very small coefficient of variation, since the uniform dispersion of the substance to be measured on the side of the small pores of the asymmetric porous membrane coincides with the concentration. The carrier-bound assay for substances according to the invention has been found to be particularly advantageous for the determination of hemoglobin or hemoglobin derivatives from whole blood. Hematocrit, the ratio of cellular components to blood volume, can also be estimated from hemoglobin concentration values.

With the use of a sufficiently hydrophilic asymmetric porous membrane, hemoglobin and methemoglobin thiocyanate, methemoglobin cyanide, methemoglobin,
Hemoglobin derivatives such as oxyhemoglobin or alkaline hematin are very highly concentrated on the side of small pores. The test carrier for measuring hemoglobin or hemoglobin derivative according to the present invention is, as an essential component, wetted with water, does not adsorb or substantially does not adsorb the hemoglobin or hemoglobin derivative to be measured, and retains a hemolytic substance. Or an asymmetric porous membrane comprising such a material in a layer on top of the membrane. This layer can be omitted if the hemolytic substance is added directly to the blood sample, such as before adding the blood sample to the test carrier.

Hemolytic agents are known to those skilled in the art. For example,
Surfactants such as anionic, cationic or non-ionic surfactants can be used in the test carriers of the present invention. Examples of anionic surfactants are sodium nonyl sulfate, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium deoxycholate, sodium dioctyl sulfosuccinate (DONS) or sodium diamyl sulfosuccinate (DANS). Cetylpyridinium chloride, cetyldimethylethylammonium bromide or cetyltrimethylammonium bromide can be used as examples of cationic surfactants. Examples of non-ionic surfactants are in particular polyoxyethylene ethers. One substance or a mixture of a plurality of substances can be used for the hemolytic agent of the present invention.

The test carrier according to the invention can either hold the hemolytic agent directly on the asymmetric porous membrane or it can be arranged in a further layer on the large pore side of the asymmetric porous membrane. If no additional layer for the hemolytic agent is used, it is advantageous to apply the hemolytic agent by impregnation to the asymmetric porous membrane. The hemolytic agent can be applied in a paste form that can be applied to the side of the large pores of the membrane and dried. If another layer is used, a suitable carrier material is likewise impregnated with a solution of the hemolytic agent or coated with a paste which can be applied with the hemolytic agent. In this case,
The type of carrier material is not important as long as the carrier material does not interfere with the measurement reaction. Possible interferences include, for example, the adsorption of hemoglobin to the carrier material or the interference reaction between the carrier material and hemoglobin or a hemoglobin derivative. For example, glass fiber fleece has been found to be suitable.

Buffer substances and substances which lead to the oxidation or complexation of hemoglobin have been found to be advantageous additives to the hemolytic agent. Buffer substances which can be considered in this respect are those whose pH value can be set at 2-12, preferably 6-8. At this point, the phosphate buffer (p
H5-8), citrate buffer (pH 2-8) and citric acid-phosphate-borate buffer (pH 2-12)
Is particularly preferred.

Any substance that oxidizes iron but does not destroy the hemoglobin structure can be used as a hemoglobin oxidizing agent. High-valent metal salts and metal complex compounds are particularly advantageous, especially potassium hexacyanoferrate (III). A substance selected from the group consisting of halides and pseudohalides can be used for complexing hemoglobin in the present method. The use of cyanide, fluoride or thiocyanate has proven to be particularly advantageous. However, the method of the invention is also suitable for detecting uncomplexed hemoglobin.

In a preferred embodiment of the test carrier used in the measurement of hemoglobin according to the present invention, the test carrier is easily and easily handled, and the finger does not touch the asymmetric porous membrane when performing the test. As such, the asymmetric porous membrane is placed on a rigid material such as a plastic foil. In this case, the asymmetric porous membrane is attached to this support so that either application or measurement of the sample to be tested takes place on the surface of the asymmetric porous membrane facing the carrier. When applying the sample to the surface of the asymmetric porous membrane facing the support, this means that the rigid support material must be able to permeate the sample. In the simplest case, the support is perforated and an asymmetric porous membrane is mounted below this hole so that the sample can be applied to the membrane through the hole. If the surface of the asymmetric porous membrane on which the measurement is made faces the support, the support should not interfere with this measurement. For this purpose, it is convenient, for example, for the support to transmit light so that optical measurements can be made. Also in this case, a hole can be in the support and the membrane can be attached to the support material above the hole so that the appropriate surface of the asymmetric porous membrane can be measured.

An advantageous embodiment of the test carrier according to the invention for the determination of hemoglobin is shown in FIGS. FIG.
Shows a test carrier that can be used to uniformly disperse the substance on the small pore side of the asymmetric porous membrane. FIG.
The test carrier consists of a strip (1) made of a rigid material such as a plastic foil with holes (4). The asymmetric porous membrane (3) attached to the strip (1) with double-sided adhesive tape (2) is placed over the hole (4). The asymmetric porous membrane (3) is placed so that the small pore side faces the strip (1). Similar test carrier compositions are disclosed, for example, in EP-A-0 256 806 or EP-A-0 407 800. However, in contrast to the prior art, the test carrier for the determination of hemoglobin according to the invention comprises a hemolytic agent and, if desired, a buffer substance and / or an oxidizing agent or complex of hemoglobin in an asymmetric porous membrane (3). It further includes substances such as forming agents. When blood (5) is added to the large pore side of the asymmetric porous membrane (3), red blood cells are lysed in the membrane (3) and released hemoglobin or the corresponding hemoglobin derivative (oxidizing agent or complexing as additional reagent) Agent is present)
Are concentrated on the small pore side of the asymmetric porous membrane (3) and spread evenly over the membrane (3). The concentration of hemoglobin or hemoglobin derivative is measured through a hole (4) on the small pore side of the asymmetric porous membrane (3).

The test carrier of FIG. 5 differs from that of FIG. 4 in that the asymmetric porous membrane (3) is attached to the strip so that the surface of the large pore side of the membrane (3) faces the strip (1). different. Blood (5) is added through the holes (4) to the large pore side of the asymmetric porous membrane (3). The measurements are taken from the small hole side of the membrane (3) opposite the hole (4). The test carrier shown in FIG. 6 has on the large pore side of the asymmetric porous membrane (3) a layer (6) impregnated with all or some of the reagents required for the test. This layer (6) contains, for example, the substances necessary to lyse the red blood cells. When blood (5) is added to the layer (6), these substances dissolve and reach the membrane (3) together with the sample liquid. The test reaction is observed from the small pore side of the membrane (3) through the hole (4).

The test carrier shown in FIG. 7 is heated to a strip (8) made of a rigid translucent material such that the asymmetric porous membrane (3) faces the strip (8) with the small pore side. Mounted by melting. For example, a layer of glass fiber fleece (6)
Is mounted on the membrane (3) in such a way that it does not come into contact with the membrane (3) but can come into contact with it by pressure from above. Therefore, at the initial position, a gap (9) exists between the film (3) and the layer (6). After adding the liquid sample to the layer (6), the sample remains there without entering the membrane (3). The residence time can be selected to a desired length. Layer (6) is membrane (3)
Layer (6) only when pressure is applied to the layer so that it can contact
Can enter the membrane (3). The small pore side of the asymmetric porous membrane (3) can be observed through the translucent strip (8). The concentration of the substance to be detected can thus be determined through the strip (8).

The present invention will be described in more detail in the following examples, but it should be understood that the present invention is not limited to these specific embodiments.

[0029]

EXAMPLES Example 1 (a) Asymmetric porous membrane (Me, Maryland, USA)
Add 10 μl of a different dye solution to the side of the large pore of BTS 25) manufactured by mtec Timonium. Preparation of dye solution Indigotin manufactured by Aldrich, Steinheim, Germany: Catalog No. 2
2.929-6) Stock solution: Dissolve 20 mg per 10 ml of water Dilution: Dissolve 10 μl of stock solution in 20 ml of water Concentration of dye solution = 1 mg / l Toluidine blue
e) (manufactured by Fluka, Buchs, Switzerland: Catalog No. 89640) a) In ethanol: Stock solution: dissolve 20 mg per 10 ml of ethanol Dilution: dissolve 100 μl of stock solution in 10 ml of ethanol Dye solution concentration = 20 mg / l b) in water 2. Stock solution: Dissolve 20 mg per 10 ml of water Dilution: Dissolve 100 μl of stock solution in 10 ml of ethanol Dye solution concentration = 20 mg / l Rhodamine B (manufactured by Aldrich, Steinheim, Germany: Catalog No. 2
5,242-5) Stock solution: 12.5 mg dissolved in 10 ml of ethanol Dilution: 100 μl of stock solution dissolved in 10 ml of ethanol Dye solution concentration = 1.25 mg / l Coomassie blue
e) (Manufactured by Serva, Heidelberg, Germany: Cat. No. CJ42655) Stock solution: 20 mg dissolved in 8 ml ethanol Dilution: 70 μl stock solution dissolved in 10 ml ethanol Concentration of dye solution = 17.5 mg / l Tartrazine (Germany,
Manufactured by Serva in Heidelberg, catalog number CJ
19140) Stock solution: 20 mg dissolved in 2 ml of water Dilution: 50 μl of stock solution dissolved in 10 ml of water Concentration of dye solution = 50 mg / l Safranin (manufactured by Aldrich, Steinheim, Germany: Catalog No. 10,
214-8) a) In ethanol: Stock solution: 20 mg dissolved in 10 ml of ethanol Dilution: 100 μl of stock solution dissolved in 10 ml of ethanol Dye solution concentration = 20 mg / l b) In water: Stock solution: 20 mg dissolved in 10 ml of water Dilution 6. Dissolve 100 μl of stock solution in 10 ml of water Dye solution concentration = 20 mg / l Acid green 41
(Manufactured by Aldrich, Steinheim, Germany: Catalog No. 21,071-4) Stock solution: 20 mg dissolved in 10 ml ethanol Dilution: 400 μl stock solution dissolved in 10 ml ethanol Dye solution concentration = 80 mg / l Bromothymol blue
blue) (E. Merc, Darmstadt, Germany)
8. Company k: Catalog No. 3026) Stock solution: 10 mg dissolved in 5 ml of pH 9 buffer Dilution: 40 μl of stock solution dissolved in 10 ml of buffer Dye solution concentration = 8 mg / l 9. Potassium hexacyanoferrate (III) (manufactured by Aldrich, Steinheim, Germany: Catalog No. 2
2,768-4) Stock solution: dissolve 20 mg in 10 ml of 0.1 N phosphate buffer, pH 3, Dye solution concentration = 2000 mg / l After adding the solution to the side of the large pore, formed on the side of the small pore Based on the coloration, it can be determined whether concentration has occurred on the side of the small pore (see table of results). (B) to test the adsorption or non-adsorption of the substance to the membrane,
Perform a filtration experiment. For this, the colored solution obtained in (a) is filtered through a plurality of membrane layers. The dye concentration in the solution before and after filtration is measured with a photometer. Measurement method Extract 2 ml from 10 ml of each dye solution,
Hewlett Packar in 0mm cuvette
d / UV spectrometer, model 845
It is measured using A as compared with a pure solvent.

BTS 25 membrane (Memtec Timnium, Maryland, USA) from 60
Cut out mm discs. 5 for each adsorption experiment
Sartorius with one membrane facing up with large holes
(Göttingen, Germany). The glass frit was previously removed from the filter holder.

The filter holder was placed on a suction bottle. The vacuum was applied and the remaining 8 ml of the dye solution was aspirated through the membrane. The filtrate was then measured in a manner corresponding to the original solution. The concentration was calculated from the absorbance. result:

[0032]

It was found that all the dyes whose concentration was reduced below 15% produced a concentration effect, whereas dyes whose concentration was further reduced did not show this effect. (C) Testing whether the test solution wets the membrane is performed as follows: one drop of solution of 20 μl in volume 15 × 15
mm on the side of the large hole of the membrane. Measure the time required for complete absorption. Repeat the experiment, placing it on the side of the smaller hole. If both times are less than 20 seconds, the solution is considered wet.

Example 2 An asymmetric porous membrane BTS 25 (Memtec Timonium, Maryland, USA) was prepared by adding 1% sodium dodecyl sulfate, 0.1N phosphate buffer, pH7.
And 0.7m mol / l K ₃ was impregnated with a solution of Fe (CN) _6. Liquid uptake was calculated as 115 ml / m ² . The membrane incorporating the liquid was dried at 50 ° C. for 30 minutes. After cooling with liquid nitrogen, the impregnated membrane was broken. The cross-section was treated with carbon vapor and used by Cambridge (Germany,
Iron was measured with a scanning electron microscope for EDX microanalysis (Nurthroho).

The iron was shown to be strongly concentrated on the small pore side of the membrane (FIG. 1). This was confirmed by visually observing the significant yellow difference between the small and large pore sides. The iron signal on the side of the large hole was obtained by visual inspection of the surface when the sample was slightly tilted in the measurement holder. Example 3 To test the concentrated dye on the small pore side of an asymmetric porous membrane, contact a BTS25 membrane (Memtec Timmonium, Maryland, USA) with each of the dye solutions of Example 1 A cross section of the membrane was prepared. This was obtained by cutting the membrane with a new razor blade. The cross section was photographed with an optical microscope equipped with a color video printer and printed as an enlarged video print.
Color prints are then reflected by a reflective optical scanner, Els
script 400 (Hirschmann, Germany)
Scanning). The average of four measurements was taken. By this method, the optical effects could be quantified. FIG. 2 shows the results for tartrazine and FIG. 3 shows the results for indigotine, taking the concentrated substance as an example. Since black is also read as a color by the scanner, the rough structure on the side of the large holes gives the impression that there is more color on the figure than actually exists.

Example 4 10 μl of cyanide methemoglobin solution (7 g / dl)
To a 12 × 8 mm asymmetric porous membrane (BTS 25, Me
mtec Timonium, Inc., Maryland, USA). After 8 seconds, the reflectance at the side of the small hole at a wavelength of 567 nm was measured. Measured 50 times and average reflectance is 4
At 0.25%, the coefficient of variation was 1.14%.

Example 5 An asymmetric porous membrane having a width of 28 cm (BTS 25, Memt)
ec Timnium, Maryland, USA)
Was impregnated with a solution of 1% by weight of sodium dodecyl sulfate, 0.1 N phosphate buffer, pH 7 and 0.7 mmol / l potassium hexacyanoferrate (III). Liquid uptake was about 115 ml / m ² . The membrane incorporating the liquid was dried at 50 ° C. for 30 minutes. 8 dried membranes
cut into strips with a width of 6 mm and a diameter of 6 m on the part where the membrane is to be attached.
It was applied to a 420 μm thick PVC foil with m holes with double-sided tape. At this time, the film is not covered with the double-sided tape at the hole. The orientation of the asymmetric porous membrane is such that the small pore side faces the pore (see FIG. 4). Approximately 10 μl of whole blood was then added to the large pore side of the membrane at the hole. The blood-stained test strip was placed in a suitable measuring device and measured 44 seconds later on the side of the small hole. A uniform dark coloration is formed on the side of the small holes between the time of sample addition and the time of measurement. Although the test apparatus did not measure any blood distribution on the sample addition side, unexpectedly high results were obtained. The coefficient of variation was 1.6% when n = 10 (n is the number of samples).

Example 6 A test strip structure similar to that of Example 5 was prepared, except that the larger hole side of the membrane faced the hole (see FIG. 5). Blood was added through a hole in the carrier foil. This somewhat simplified the accuracy of sample addition. The measurement was performed from the side of the small hole. The results were the same as in Example 5.

Example 7 Example 5 differs in that the impregnating solution has the following composition:
A test strip with a structure similar to (corresponding to FIG. 4) was prepared: 0.3% by weight sodium dioctylsulfosuccinate (DONS) (E. Merck, Darmstadt, Germany) 0.3% by weight sodium diamylsulfosuccinate (D
ANS) (Cyanamid, Wolfratshausen, Upper Bavaria, Germany) 0.2% by weight Saponin 0.2% by weight Potassium hexacyanoferrate (III) (0.1 mol / l citrate buffer, pH 6.8)
Middle) The measurement was performed in the same manner as in Example 5. In this case, n = 10 and the coefficient of variation was 1.25%.

Example 8 Transparent 200 μm thick foil (Pokalon, Weil am Main Lonza, Germany) and glass fiber fleece (Trapo 83/14, JC Binzer)
Asymmetric Porous Membrane (BTS 25) as a test strip between Co., Ltd., Hatsfeld / Edel, Germany.
At this time, the side of the large hole faces the glass fiber fleece, but the membrane and the glass fiber fleece are not in contact with each other (see FIG. 7). The glass fiber fleece was impregnated with methemoglobin cyanide. After completion of the impregnation, the test strip was measured by reflectance photometry at a wavelength of 567 nm in a suitable device. In this measurement method, the impregnated glass fiber fleece and the asymmetric membrane were brought into mechanical contact before the measurement was started by the measurement device. 7g methemoglobin cyanide concentration
At / dl, a reflectivity of 32.69% was observed. The coefficient of variation was 1.77%.

Example 9 Except for the membrane between the glass fiber fleece and the transparent foil,
The experiment of Example 8 was repeated. As a result, the coefficient of variation was 5.84%. Examples 8 and 9 clearly suggest that the use of a membrane leads to a more uniform color reproduction. This method is not limited to analytical methods for measuring hemoglobin in blood, but can be applied in all cases where it is necessary or desirable to increase the uniformity of the coloring.

Example 10 Bromothymol blue was measured in the same manner as in Example 3 as a substance not concentrated on the small pore side of the asymmetric porous membrane.
Impregnated and dried BT after contact with the corresponding dye solution
S25 membrane (Memtec Timmonium, Maryland, USA) was treated with liquid paraffin (melting point 56-5).
8 ° C, E.C. (Merck, Darmstadt, Germany) and then lifted. The membrane was thus coated with a thin paraffin layer. The paraffin layer stabilizes the structure but does not dissolve the dye or the film itself.

An enrichment profile was prepared as in Example 3. FIG. 8 shows the coloring intensity, showing that the dye concentration is substantially constant throughout the film thickness.

[Brief description of the drawings]

FIG. 1: Scanning electron E showing a cross section of an asymmetric porous membrane impregnated with K ₃ [Fe (CN) ₆ ] treated with carbon vapor
FIG. 3 shows a diagram of DX microanalysis.

FIG. 2 shows the coloring intensity of a section of an asymmetric porous membrane impregnated with tartrazine by reflectance photometry.

FIG. 3 shows the coloring intensity of a section of an asymmetric porous membrane impregnated with indigotin by reflectance photometry.

FIG. 4 shows a sectional view of a preferred embodiment of the test carrier according to the invention.

FIG. 5 shows a sectional view of a preferred embodiment of the test carrier according to the invention.

FIG. 6 shows a sectional view of a preferred embodiment of the test carrier according to the invention.

FIG. 7 depicts a cross-sectional view of a test carrier showing the material uniformly dispersed on the small pore side of the asymmetric porous membrane.

FIG. 8 is bromothymol blue.
1 blue) shows the coloring intensity of the cross section of the asymmetric porous membrane impregnated by the reflectance photometry.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hans Lange, Germany D-6840 Ramper Thyme Römer-Strasse 99 D (72) Inventor Manfred Zeiler, Germany D-6822 Alt Scheim Mühlstraße 52 (56 References JP-A-3-56856 (JP, A) JP-A-2-268819 (JP, A) JP-A-55-126855 (JP, A) JP-A-1-202661 (JP, A) (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/52 G01N 33/72 G01N 31/22

Claims (9)

(57) [Claims] 1. A membrane-wetting solution of a substance, comprising contacting one side of the membrane and the interior thereof with an asymmetric porous membrane that does not or substantially does not adsorb the substance .
A method of uniformly dispersing and concentrating a substance in a part . 2. The asymmetric porous membrane has an asymmetry factor of 1
The method of claim 1, wherein the value is greater than zero. 3. The method according to claim 1, wherein a solution of the substance to be measured is brought into contact with an asymmetric porous membrane, or the substance to be measured in the sample is present in a layer preceding the membrane. In the liquid sample, which is formed or released by one or more substances, and is measured on the side of the small pores, wherein the substance to be measured is not adsorbed or substantially not adsorbed on the membrane. Carrier-binding assay for the substance 4. The asymmetric porous membrane has an asymmetry factor of 1
4. The method of claim 3, wherein the value is greater than zero. 5. The pore size on the small pore side of the asymmetric porous membrane is about 0.003-3 μm.
The method described in. 6. The method according to claim 3, wherein the substance to be measured is hemoglobin or a hemoglobin derivative. 7. An asymmetric porous membrane which does not adsorb or substantially does not adsorb hemoglobin and which itself retains or contains a hemolytic substance in a layer preceding it. A test carrier for performing the method of claim 6, comprising a test carrier. 8. The asymmetric porous membrane further retains a substance that leads to free hemoglobin oxidation or complex formation.
A test carrier according to item 1. 9. The asymmetric porous membrane is mounted on a solid material such that the addition of the substance to be measured in the sample or the measurement of the substance in the sample is performed on the side of the asymmetric porous membrane facing the solid material. The test carrier according to claim 7.